1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using a promoter naturally associated with a Zea mays nuclear gene encoding a chloroplast localized fructose-1,6-bisphosphate aldolase.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Paszkowski et al., 1984; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), Ti plasmid nopaline synthase (nos, Ebert et al., 1987), alcohol dehydrogenase (Adh, Walker et al., 1987), and sucrose synthase (Yang and Russell, 1990).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991), α-tubulin (Carpenter et al., 1992; Uribe et al., 1998), cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), and chalcone synthase promoters (Franken et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
Fructose-1,6-bisphospate (F16BP) aldolase (International Classification of Enzymes No. 4.1.2.13) is a gene which is expressed in an inducible constitutive manner in maize. This enzyme is essential in carbon metabolism and participates in the Calvin cycle, gluconeogenesis and glycolysis. F16BP aldolase catalyzes the conversion of fructose-1,6-bisphospate to dihydroxyacetone phosphate and 3-phosphoglyceraldehyde in a reversible manner. Antisense inhibition of potato aldolase activity has been found to result in a decrease in the rate of photosynthesis, a decrease in starch synthesis and an overall reduction in growth (Haake et al., 1998; Haake et al., 1999).
Two distinct classes of aldolases have been identified (Rutter 1964; Marsh and Lebherz, 1992; Gross et al., 1994; Pelzer-Reith et al., 1994). Class I aldolases are found predominantly in higher plants, animals, ferns, mosses and some eukaryotic algae. These aldolases function as tetramers and can be found in the cytoplasm or plastids, preferably chloroplasts, in the cell. The genes encoding aldolases localized to either the cytoplasm or the plastids, particularly the chloroplasts, are encoded by genes residing in the nuclear genome (Anderson and Levin, 1970; Lebherz et al., 1984; Kayaga et al., 1995). Herein, aldolases localized to the chloroplasts are referred to as chloroplastic aldolases. Class II aldolases are typically found in bacteria, cyanobacteria, fungi and some algae. Class II aldolases function as a dimer and can also be found in either the cytoplasm or plastids of the cell. The two classes of aldolases can be further distinguished as Class I types form a Schiff-base with the substrate and are not inhibited by EDTA; in contrast, Class II types do not form a Schiff-base and can be inhibited by EDTA (Rutter, 1964).
C3 and C4 plants differ greatly in their initial fixation of CO2 and in their physical compartmentalization of carbon metabolism. In the chloroplasts of C3 plants (such as rice), ribulose-1,5-bisphosphate is the acceptor molecule for CO2 and the 6 carbon intermediate is catalyzed to 2 molecules of 3-phosphoglycerate. Both carbon fixation and subsequent use of the products in the Calvin cycle is carried out in the chloroplasts of mesophyll cells. In contrast, C4 plants (such as maize) fix CO2 into four-carbon acids such as malate. The fixed carbon is then transported from the mesophyll cells into the bundle sheath cells which surround the vascular tissues. It is in the bundle sheath cells where the fixed CO2, carried as malate, is released and metabolized via the Calvin cycle. F16BP aldolase is an essential enzyme in the Calvin cycle in both C3 and C4 plants. However, since these two plant types vary significantly in their compartmentalization and regulation of carbon metabolism, it is unknown whether mechanisms regulating carbon metabolism will show similar control functions in C3 and C4 plants.
F16BP aldolases have been reported from a number of plant sources including spinach (Kruger and Schnarrenberger, 1983; Lebherz et al., 1984; Pelzer-Reith et al., 1993), potato (Haake et al., 1998; Haake et al., 1999), rice (Hidaka et al., 1990; Tsutsumi et al., 1994; Schaeffer et al., 1997), Arabidopsis (Chopra et al., 1990) and maize (Gasperini and Pupillo, 1982/3; Kruger and Schnarrenberger, 1983; Kelly and Tolan, 1986). The complexity of the aldolase encoding genes vary from versions with a single intron in maize (Dennis et al., 1988) to those with 5 introns in rice (Tsutusmi et al., 1994). The genes appear to be single to low copy in several species examined (Pelzer-Reith et al., 1993; Tsutsumi et al., 1994).
In rice, which is a C3 monocot, genomic clones for both the cytoplasmic and chloroplastic F16BP aldolases have been isolated (Hidaka et al., 1990; Tsutsumi et al., 1994; Nakamura et al., 1996). A published AldP chloroplast genomic sequence discloses 182 base pairs upstream of the initiating ATG (GenBank Accession number D13513; Tsutsumi et al., 1994). A published AldC-a cytoplasmic genomic sequence discloses 211 bp upstream of the initiating ATG (GenBank Accession number D13512; Tsutsumi et al., 1994). In young seedlings, aldolase was found to be induced by oxygen deprivation (Umeda and Uchimiya, 1994). The chloroplastic version of aldolase was found to be expressed mainly in the mesophyll cells in the leaf blade and to be light-responsive (Nakamura et al., 1997). When the rice chloroplastic aldolase promoter was fused with the uidA reporter gene and transformed into tobacco (a C3 dicot), GUS expression was found mainly in the leaves (Kayaga et al., 1995).
In maize, a C4 monocot, only the nuclear gene encoding the cytoplasmic form of F16BP aldolase has been cloned (Kelly and Tolan, 1986; Dennis et al., 1988) and this version of the enzyme has been localized to mesophyll cells (Taylor, 1989). Although the nuclear gene encoding the chloroplastic version of maize aldolase has not bee cloned, the protein product has been localized to the bundle sheath cells in leaves (Kruger and Schnarrenberger, 1983). Because of the marked differences in C3 and C4 carbon metabolism, it is unknown whether an aldolase promoter from a C4 type plant will show the same expression pattern or regulation as an aldolase promoter from a C3 type plant.
Although the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter sequences with beneficial expression characteristics. The number of effective promoters available for use with transgenes in maize is not abundant. New promoters, especially promoters that will express differentially in maize tissues, are spatially and/or temporally expressed, show proper regulation with regard to essential metabolic processes, or are induced to express by different environmental signals, would be useful. Such expression specific promoters could be useful in minimizing yield drag and other potential adverse physiological effects on maize growth and development that might be encountered by high-level, non-inducible, constitutive expression of a transgenic protein in a plant. A wider range of effective promoters also would make it possible to introduce multiple transgenes into a plant, each fused to a different promoter, thereby minimizing the risk of DNA sequence homology dependent transgene inactivation (co-suppression). Therefore, there is a great need in the art for the identification of novel inducible promoters which can be used for the high-level expression of selected transgenes in economically important crop plants.